Salmonella enterica serovar Typhi causes typhoid fever, a severe infection that affects more than twenty million people every year. Much knowledge has been gained over the last twenty years concerning how Salmonella causes disease through manipulating host cellular pathways and how host cells respond. However, there is tremendous variation in who gets infected and the severity of infections. How host signaling pathways and responses are affected by human genetic variation to modulate susceptibility to Salmonella infection is poorly understood. This naturally occurring host genetic variation is an untapped resource that could reveal novel components of pathways, prediction of susceptibility, and possibly host-directed therapeutic targets. The long-term goal of this research is to understand how host genetic differences alter the responsiveness and susceptibility of cells to Salmonella infection and how this affects risk and severity of disease in people. In pursuit of this goal, genome-wide association studies (GWAS) of cellular Salmonella infection traits were carried out using a platform called Hi-HOST (High-throughput Human in vitro Susceptibility Testing). In Hi- HOST screens, precise measurements of infection readouts are conducted on cells derived from hundreds of genotyped individuals. Genome-wide association is then used to identify genetic differences that underlie variation in the cellular infection phenotypes. Using Hi HOST, three new regulators that affect Salmonella- induced cell death in cells and sepsis in human populations were identified and characterized. The objective of this application is to use Hi-HOST and subsequent studies to define and characterize host genetic differences that alter S. typhi invasion and early survival. The host genetic differences serve as the starting point for mechanistic studies to determine how host pathways involving macropinocytosis and early survival are altered. By integrating these cellular studies with animal models and genotyping of human typhoid fever cohorts, an in depth understanding will be achieved for how genetic differences contribute to who gets sick and why. In Aim 1, common human genetic variation that modulates S. typhi invasion will be identified and validated. In Aim 2, the impact of variation in S. typhi invasion on typhoid fever in human populations and Salmonella infections in animal models will be assessed. In Aim 3, mechanisms for how the newly identified regulators modulate S. typhi invasion will be characterized. Carrying out these Aims will result in the elucidation of new mechanisms in host-pathogen interactions and ultimately a broad understanding for how genetic differences determine which individuals are at risk for typhoid fever and possibly other diseases caused by pathogens which utilize similar invasion mechanisms. With rising concerns over resistance to broad-spectrum antimicrobials, this study may leverage our genetic differences to reveal new therapeutic targets focused instead on host modulation.